Demand is exceedingly great for sweetening agents which impart minimal caloric value to the foods in which they are contained. Low or noncaloric sweeteners are the key ingredient in low-calorie diets, and are particularly important for persons who are overweight, who are diabetics, or who are particularly susceptible to dental caries. Artificial sweeteners account for $1 billion of the $3.5 billion U.S. food additive market. Several well known compounds have been used, and continue to be used to some extent, as artificial sweeteners including saccharin, cyclamate, acesulfame K (ACK) and aspartame (which is a dipeptide). Aspartame, saccharin and ACK are approved for use in foods in the United States and other compounds such as sucralose and alitame are awaiting approval by the FDA.
In addition to these small molecules, two naturally occurring proteins have been suggested as low-calorie sweeteners, since they are many times sweeter than sugar. Thaumatins I and II are marketed under the brand name Talin.RTM. and are obtained from the fruits of the West African plant Thaumatococcus daniellii. Thaumatin is a nontoxic, noncarcinogenic protein that has a sweetness between 1600 and 3000 times that of sucrose on a weight basis. The other naturally occurring protein, monellin is derived from the "Serendipity Berry" and is 1500-3000 times sweeter than sugar. Both thaumatin and monellin denature at high temperatures, but monellin has been produced in a single-chain form and the single-chain form resists denaturation. See, for example, U.S. Pat. No. 5,234,834.
These two proteins and an additional protein, "brazzein" isolated from Pentadiplandra brazzeana Baillon have been manipulated recombinantly. See U.S. Pat. No. 5,346,998 (brazzein), PCT publications WO 85/01746 and WO 87/03007, and U.S. Pat. Nos. 4,771,000; 4,891,316; 4,966,842; and 5,221,624 (thaumatins) and EP 374157 B1 (single-chain monellin). Refolding of recombinant thaumatin has been described in European patent EP 255823 B, and single-chain monellin has been produced in transgenic plants to confer inherent sweetness (PCT application WO 92/01790). A yeast expression system for single-chain monellin has also been described in WO 90/07580.
The sweetener protein that forms the subject of the present invention, mabinlin (MBL), is derived from the seeds of Capparis masaikai. It is a heterodimer and exists in at least five isoforms. The complete amino acid sequence of one of these isoforms, mabinlin II (MBLII), was described by Liu, X. et al. Eur J Biochem (1993) 211:281-287. According to this article, the A chain of mabinlin II contains 33 amino acids; the B chain contains 72. The article shows that MBLII has considerable homology with a 2S seed storage protein 3(AT2S3) derived from A. thaliana. The dipeptide is water-soluble and is approximately 400 times sweeter than sucrose. Despite being a heterodimer, it exhibits high heat stability and is still sweet after 48 hours at 85.degree. C.
The complete amino acid sequence of three other isoforms, I-1, III and IV were reported by Nirasawa et al., Eur J Biochem (1994) 223:989-995. As described by this article, there is a high degree of sequence identity among the amino acid sequences of these isoforms. MBLIII and IV, like MBLII, are heat stable while MBLI-1 is sensitive to high temperature treatment; i.e., loss of sweet activity after 1 hour incubation at 80.degree. C.
The present invention provides recombinant materials for the production of mabinlin in practical amounts and for the production of transgenic plants containing inherently sweet edible parts by virtue of production of mabinlin in situ. Furthermore, production of single-chain mabinlin is provided.